Titin, a giant protein that spans the half sarcomere, functions as a molecular spring that underlies the passive and restorative forces that maintain the structural integrity of the contracting sarcomere. The contribution of titin to passive muscle stiffness has been well studied in cardiac muscle but in contrast titin is poorly understood in skeletal muscle. Due to the many differences between skeletal and cardiac muscle, separate studies are needed for each muscle type. Passive stiffness of skeletal muscle greatly influences functional activities and quality of life, and, thus, the molecular mechanisms of passive stiffness generation and the role of titin in this stiffness need to be studied. We will first perform stiffness measurements at several levels of complexity (from skinned fibers to whole muscle) and determine the contribution of titin to passive stiffness of each. We will investigate how titin's contribution to passive stiffness varies in different muscles, depending on differential splicing and posttranslational modifications in titin. To facilitate the speed and accuracy in determining the sequence of titin isoforms expressed in different muscle types, we developed a titin exon microarray which contains all of titin's exons found in a range of species (363 exons in human). We anticipate that our work will greatly increase the understanding of the role of titin in passive muscle stiffness and that this will provide a sound basis for understanding its role in muscle diseases, which we will address next. We will establish titin's stiffness in over-load induced hypertrophy and disuse-induced atrophy, relative to the changes in the extracellular matrix (collagen and elastin). Titin's role in disease will also be studied in tenascin-X (TNX) deficient patients (one of the types of Ehlers-Danlos syndrome) and TNX KO mice; our preliminary data indicate increased titin-based stiffness, as compensatory response to counteract the decreased collagen- stiffness of TNX-deficient muscle. Our last aim will critically test the proposal that titin functions as a biomechanical sensor that triggers hypertrophy. The main focus will be on a genetically engineered mouse model that is deficient in PEVK exons 219-225 (PEVK KO). (The PEVK is an important source of elasticity of the titin spring). Preliminary data show that skeletal muscles of the PEVK KO are significantly hypertrophied, and we will investigate the signaling pathways involved. We will use a candidate approach, including a study of the interaction between titin and the mTOR signaling pathway (this pathway has previously been shown to increase protein synthesis in response to stretch of passive muscle) and of the role of titin-binding proteins previously linked to hypertrophy signaling and highly upregulated in the PEVK KO. We will dissect their roles in hypertrophy signaling by crossing the PEVK KO with models in which these proteins have been deleted. Understanding the mechanisms that regulate muscle hypertrophy is clinically important because loss of mass is often a consequence of diseases and it debilitates the elderly and bedridden patients. Overall, the proposed work will be a major step towards our long-term goal, which is to gain a detailed understanding of the roles of titin in skeletal muscle structure and function, in health and disease.